Lateral diffusion of proteins in the periplasm of Escherichia coli

Brass, J. M.; Higgins, Christopher F.; Foley, Michael; Rugman, P A; Birmingham, John J.; Garland, P. B.

doi:10.1128/jb.165.3.787-795.1986

Cited by 102 publications

(61 citation statements)

References 57 publications

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“…Interestingly, the maltose-binding protein (MBP), which is associated with the chemoreceptor Tar to sense maltose, also exhibits a polar localization (14). In addition, MBP can diffuse laterally in the periplasm (36), and the induction of its synthesis by adding maltose in the medium triggers polar cap formation in E. coli (37). These observations strongly suggest that bacteria detect the nutrient substances at the poles.…”

Section: Discussionmentioning

confidence: 99%

Translocation of Jellyfish Green Fluorescent Protein via the Tat System of Escherichia coli and Change of Its Periplasmic Localization in Response to Osmotic Up-shock

Santini¹,

Bernadac²,

Zhang³

et al. 2001

Journal of Biological Chemistry

180

121

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The bacterial twin arginine translocation (Tat) pathway is capable of exporting cofactor-containing enzymes into the periplasm. To assess the capacity of the Tat pathway to export heterologous proteins and to gain information about the property of the periplasm, we fused the twin arginine signal peptide of the trimethylamine N-oxide reductase to the jellyfish green fluorescent protein (GFP). Unlike the Sec pathway, the Tat system successfully exported correctly folded GFP into the periplasm of Escherichia coli. Interestingly, GFP appeared as a halo in most cells and occasionally showed a polar localization in wild type strains. When subjected to a mild osmotic up-shock, GFP relocalized very quickly at the two poles of the cells. The conversion from the halo structure to a periplasmic gathering at particular locations was also observed with spherical cells of the ⌬rodA-pbpA mutant or of the wild type strain treated with lysozyme. Therefore, the periplasm is not a uniform compartment and the polarization of GFP is unlikely to be caused by simple invagination of the cytoplasmic membrane at the poles. Moreover, the polar gathering of GFP is reversible; the reversion was accelerated by glucose and inhibited by azide and carbonyl cyanide m-chlorophenylhydrazone, indicating an active adaptation of the bacteria to the osmolarity in the medium. These results strongly suggest a relocalization of periplasmic substances in response to environmental changes. The polar area might be the preferential zone where bacteria sense the change in the environment.The periplasmic space lies between the inner and the outer membranes of Gram-negative bacteria. A number of processes that are vital to the growth and viability of the cell occur within this compartment. Proteins residing in the periplasmic space fulfill important functions in the detection, processing, and uptake of essential nutrient substances. These proteins are exported into the periplasm mainly via two pathways: the unfolded proteins via the Sec system (1) and the folded enzymes containing redox cofactor via the Tat 1 (or Mtt) pathway (2-4).The periplasm might not be a uniformly homogenous compartment; fine structures known as Bayer patches/bridges (5) and periseptal and polar annuli (6 -9) have been described. The existence of these structures under physiological conditions is a subject of contention (10,11). Nevertheless, these structures were proposed to provide sites required for the export of outer membrane components, murein synthesis, secretion of bacteriophages, and cell divisions (12).On the other hand, polar bacterial organization was observed with a variety of bacterial species and concerns a disparate array of cellular functions (13). In addition to the well known examples of polar organelles such as flagella, pili, and stalklike appendages at the bacterial surface, accumulating evidence shows that periplasmic, inner membranous, and cytoplasmic proteins may also exhibit polar localization under certain condition. These proteins participate in various cellula...

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Section: Discussionmentioning

confidence: 99%

Translocation of Jellyfish Green Fluorescent Protein via the Tat System of Escherichia coli and Change of Its Periplasmic Localization in Response to Osmotic Up-shock

Santini¹,

Bernadac²,

Zhang³

et al. 2001

Journal of Biological Chemistry

180

121

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“…It seemed possible that labeled proteins might penetrate only into the periplasmic space and that plasmolysis might result in expansion of the periplasm into what are normally cytoplasmic regions of the cell, thus giving the appearance of cytoplasmic labeling. Plasmolyzed cells grossly resembling the triplet distribution have been described (1). We also considered the possibility that the nucleoid might quench fluorescence or otherwise perturb fluorescent emissions emanating from the cell surface or the periplasmic space.…”

Section: Methodsmentioning

confidence: 99%

“…Once in the periplasm, DNA can be transferred into the cell interior by an unknown mechanism; however, proteins do not get past the cytoplasmic membrane in Ca2+-permeabilized cells. These properties have been used to introduce proteins selectively into the periplasmic space (1). As early as 1958, it was observed that the envelope of gram-negative bacteria could be altered in its permeability by brief exposure to EDTA (22,28).…”

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confidence: 99%

Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli

Shellman

Pettijohn

1991

J Bacteriol

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Growing bacterial cells forming division septa have sites near the septa that are sensitive to EDTA shock. Cells treated with EDTA incorporate proteins and other molecules from the surrounding medium, probably via vesiclelike lesions at the septa that are induced by EDTA. The amount of protein taken up is proportional to the protein concentration in the permeabilization medium. Incorporated molecules equilibrate throughout the cytoplasm, and those with affinity for DNA bind to the nucleoid. Conditions that promote the viability of permeabilized cells and help to avoid otherwise irreversible effects of EDTA are defined. Procedures for selecting cells that have incorporated protein and for studying the distribution of the protein and its effects in growing-dividing cells are described. The procedure may have several applications to molecular and cellular biology; however, we describe here the localization in living cells of the histonelike protein HU. Fluorescence microscopy of cells containing different amounts of fluorescein-labeled HU (varied from approximately 10(3) to 10(5) molecules per cell) showed that the HU concentrates in the nucleoid and is uniformly distributed throughout this structure. Control experiments demonstrated that unlabeled interior parts of the nucleoid can be resolved when labeled proteins that do not bind DNA or enter the nucleoid are introduced into living cells. It was concluded that in vivo added HU binds primarily DNA and that there are no intrinsic restrictions on major regions of the nucleoid to which the added HU protein may bind.

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“…By monitoring the kinetics and the extent of movement of fluorescent molecules from the unbleached area into the bleached area, both the percentage of fluorescent molecules that are mobile and their diffusion coefficient can be calculated. This technique has been used successfully to measure the movement of a number of macromolecules in bacteria (21)(22)(23)(24)(25). The fluorescent molecule we chose for use was the GFP from Aequoria victoria, because this protein and its many variants have been used to measure the mobility of soluble proteins in a number of systems (26)(27)(28).…”

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confidence: 99%

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

Cowan

Koppel²,

Setlow³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

146

122

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Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of Bacillus subtilis but becomes freely mobile in germinated spores in which cytoplasmic water content has increased Ϸ2-fold. The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated cwlD spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other Bacillus species. The immobility of a normally mobile protein in dormant wild-type spores and germinated cwlD spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of Bacillus species.S pores of various Bacillus species are formed in the process of sporulation, and these spores are adapted for long-term survival because they are resistant to many environmental stresses and are metabolically dormant (1-4). However, given the proper stimulus, generally the appearance of specific nutrients in the environment, the dormant spores can return to life through the process of spore germination and then outgrowth (2). A major factor in spore dormancy and resistance is the low level of water in the spore cytoplasm or core [25-55% of the mass of the hydrated dormant spore core depending on the species (1, 3-5)]; another factor may be the high level of pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)] (Ϸ10% of the mass of the hydrated stage II-germinated spore core) in the spore core, likely present as a chelate with divalent cations, predominantly Ca 2ϩ (1,6). DPA is released in the first minutes of spore germination and is replaced with water as the spore-core water content rises slightly; these spores are said to have completed stage I of germination (2, 7). Subsequently, the large peptidoglycan cortex around the spore core is degraded, and removal of this restraint allows the spore core to expand rapidly by uptake of water and thus complete stage II of germination (2, 7). This latter event results in a level of core water (75-80% of wet weight) that is similar to that in growing cells (8). As noted above, there is neither metabolism nor enzyme action in the cytoplasm of dormant spores, although there are several enzyme-substrate pairs located in this region of the spore (3). There is also no detectable metabolism or enzyme action in the core of stage I-germinated spores that have excreted their DPA but have not degraded their cortex (7). However, in fully germinated (stage II) spores in which the cortex has been degraded and the cytoplasm is fully hydrated, enzyme action and metabolism begin rapidly (2).One explanation that has been put forward for the lack of enzyme action in dormant and stage I-germinated spores is that the level of water in these spores is too low to allow enzyme a...

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Lateral diffusion of proteins in the periplasm of Escherichia coli

Cited by 102 publications

References 57 publications

Translocation of Jellyfish Green Fluorescent Protein via the Tat System of Escherichia coli and Change of Its Periplasmic Localization in Response to Osmotic Up-shock

Translocation of Jellyfish Green Fluorescent Protein via the Tat System of Escherichia coli and Change of Its Periplasmic Localization in Response to Osmotic Up-shock

Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

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